The present invention concerns the treatment of nerve damage with the Insulin-like Growth Factor I (IGF-I) isoform known as mechano growth factor (MGF). More particularly, MGF is localised around the sites of such damage to effect repair, typically by means of the placement of a conduit around the two ends of a severed peripheral nerve,
IGF-I and MGF
Mammalian IGF-I polypeptides have a number of isoforms, which arise as a result of alternative mRNA splicing. Broadly, there are two types of isoform, liver-type isoforms and non-liver ones. Liver-type isoforms may be expressed in the liver or elsewhere but, if expressed elsewhere, are equivalent to those expressed in the liver. They have a systemic action and are the main isoforms in mammals. Non-liver isoforms are less common and some are believed to have an autocrine/paracrine action. A cDNA of the latter type has been cloned, as discussed below, following detection in skeletal and cardiac muscle undergoing mechanical overload.
The terminology for the IGF-I splice variants is based on the river isoforms (Chew et al, 1995) and has not fully evolved to take into account those produced by non-liver tissues The latter are controlled to some extent by a different promoter (promoter 1) to the liver IGP-I isoforms, which respond to hormones and are under the control of promoter 2 (Layall, 1996).
For the purposes of this invention, two isoforms are of particular interest. These are both expressed in skeletal muscle, though it has only recently been appreciated that two muscle isoforms exist. The first isoform is muscle liver-type IGF-I or L.IGF-I (systemic type), which is of interest mainly for comparative purposes. The second is mechano-growth factor or MGF (autocrine/paracrine type).
These are alternative splice variants. Exons 1 and 2 are alternative leader exons (Tobin et al, 1990; Jansen et al, 1991) with distinct transcription start sites which are differentially spliced to common exon 3. Exons 3 and 4 code for the mature IGF-I peptide (B, C, A and D domains) as well as the first 16 amino acid of the E domain. Exons 5 and 6 each encodes an alternative part of a distinct extension peptide, the E domain. This is followed by the termination codons of precursor IGF-I, 3xe2x80x2 untranslated regions and poly(A) addition signal sites (Rotwein et al, 1986). A further difference between the two isoforms is that MGF is not glycosylated and is therefore smaller. It has also been shown to be less stable. It may thus have a shorter half-life.
It has been shown that MGF, which is not detectable in skeletal muscle unless it is subjected to exercise or stretch (Yang et al, 1996), has exons 4, 5 and 6 whilst the muscle L.IGF-I has exons 4 and 6. Exon 5 in MGF has an insert of 52 bp which changes the 3xe2x80x2 reading frame and hence the carboxy end of the peptide In addition, MGF has been detected in overloaded cardiac muscle (Skarli et al, 1998).
Functional epitope mapping of IGF-I using a battery of monoclonal antibodies (Ma{haeck over (n)}es et al, 1997) has shown that the carboxy terminus (3xe2x80x2 end) of IGF-I is important in determining the affinity of the peptide for a particular receptor and/or binding protein.
MGF mRNA is not detected in dystrophic muscle even when it is subjected to stretch. The inability of muscle in both the autosomal- and dystrophin-deficient dystrophies to respond to overload by stretch (Goldspink et at, 1996) indicates that the cytoskeleton may be involved in the transduction mechanism. It is probable that there is a basic mechanism that detects muscle overload and which results in the expression of both variant forms of IGF.
Thus, MGF is known to be expressed in skeletal and cardiac muscle tissue in response to stretch and exercise and as a result is believed to be involved in repair of damage to muscle (Yang et al, 1996; WO97/33997). This has been confirmed more recently by McKoy et at (1999).
Conduits
It has previously been proposed to use a conduit to assist in nerve damage repair, e.g. to bridge a gap in a severed nerve. The aim is to place the conduit around the nerve, e.g. around its two severed ends, so that the nerve will regrow within the conduit.
In particular, conduits composed of Poly-3-hydroxy-butyrate have been proposed as an alternative to nerve autografts, which result in sub-optional functional results and donor site morbidity. PHB occurs within bacterial cytoplasm as granules and is available as bioabsorbable sheets. PHB conduits have been shown to assist in nerve regeneration and to show good results compared to nerve autografts (Hazari et al, J. Plastic Surgery (1999)).
Various different conduit materials have been proposed, including PHB, but none have yet been fully applied clinically. Only silicone has been applied, in a restricted clinical trial (Lundborg et al, 1997), but a second operation has sometimes been necessary to remove the non-resorbable silicone tube.
We have now identified a new and surprising property of MGF.
Plasmids containing MGF DNA operably linked to expression signals capable of securing expression in muscles were prepared and injected intramuscularly into rats. Expression of MGP in vivo resulted. To investigate the effect of MGF on the animal""s nerves, the right-facial nerve was damaged by avulsion in some animals and crushing in others. Similar experiments were performed with plasmids capable of expressing L.IGF-I and control experiments were also carried out using equivalent xe2x80x9cemptyxe2x80x9d plasmids lacking an MGF or L.IGF-I coding sequence, and with non-operated rats.
The surgical procedures carried out normally result in massive motoneurone loss, and that was the case in the control animals. However, in the case of nerve avulsion, use of L.IGF-I reduced motoneurone loss to about 50% and use of MGF reduced motoneurone loss to about 20%. Although both isoforms were found to be effective in promoting motoneurone rescue, MGF was, surprisingly, more than twice as effective as L.IGF-I. This opens up the possibility of using MGF in the treatment of neurological disorders, especially motoneurone disorders. Additionally, it should be noted that this is the first time that altered availability of neurotrophic factors to intact adult motoneurones has been shown to affect a subsequent response to injury and also that this is the first time that intramuscular gene transfer using plasmid DNA has been shown to be an effective strategy for motoneuronal rescue.
IGF-I isoforms have specific binding proteins which determine their action, particularly in terms of which tissues the isoform takes effect in. It appears that the binding protein for MGF is located in the central nervous system (CNS) as well as in skeletal and cardiac muscle. This may explain its greater effectiveness. Also, the fact that MGF is not glycosylated and thus smaller than L.IGF-I may facilitate its transfer from the muscle to the motor neuron cell bodies in the CNS.
These findings have general applicability to the treatment of neurological disorders and are surprising because MGF had previously only been detected in cardiac muscle and skeletal muscle under stretch/exercise. Chew (1995) suggests that an IGF-I Ec form is found in the liver. However, this is detectable in very low amounts and may be due to leaky transcription. Therefore, it had previously been believed that MGF was a muscle-specific isoform whereas it has now emerged that it is also implicated in repairing damage to the nervous system and can thus form the basis of treatments for disorders of the nervous system.
Moreover, our findings show that MGF will be useful in repairing nerve damage, especially in the peripheral nervous system (PNS), when localised around the site of the damage. In particular, MGF will be useful in repairing nerve damage in conjunction with a conduit placed around the two ends of a severed nerve. Notably, we have found that, by placing the two ends of a severed rat sciatic nerve in juxtapostion in a conduit and filling with a gel comprising a vector containing MGF cDNA, repair of a 3 mm gap in the nerve was achieved in as little as two weeks. The properties of MGF in nerve regeneration, as identified by the present Inventors, can be combined with the tendency of such conduits to facilitate nerve regeneration. This will result in an improved conduit-based means of repairing nerve damage. Other means of localising MGF at the site of damage can also be used.
Accordingly, the invention provides:
a method of treating nerve damage comprising administering to a subject in need thereof an effective non-toxic amount of an MGF (mechano-growth factor) Insulin-like Growth Factor I (IGF-I) isoform comprising amino acid sequences encoded by nucleic acid sequences of IGF-I exons 4, 5 and 6 in the reading frame of MGF and having the ability to reduce motoneurone loss by 20% or greater in response to nerve avulsion, by localisation of said MGF at the site of said damage.
The invention also provides:
a kit for the treatment of nerve damage comprising:
(a) an MGF IGF-I isoform of the invention; and
(b) a conduit of the invention; and optionally
(c) a polypeptide growth factor which prevents or diminishes degeneration; and optionally
(d) another neurologically active agent.